Exploration of the prediction and generation patterns of heterocyclic aromatic amines in roast beef based on Genetic Algorithm combined with Support Vector Regression.

Heterocyclic aromatic amines (HAAs) are harmful byproducts in food heating. Therefore, exploring the prediction and generation patterns of HAAs is of great significance. In this study, genetic algorithm (GA) and support vector regression (SVR) are used to establish a prediction model of HAAs based on heating conditions, reveal the influence of heating temperature and time on the precursor and formation of HAAs in roast beef, and study the formation rules of HAAs under different processing conditions. Principal component analysis (PCA) showed that the effect on HAAs generation increases with the increase of heating temperature and time. The GA-SVR model exhibited near-zero absolute errors and regression correlation coefficients (R) close to 1 when predicting HAAs contents. The GA-SVR model can be applied for real-time monitoring of HAAs in grilled beef, providing technical support for controlling hazardous substances and intelligent processing of heat-processed meat products.

Comparing application methods of reishi mushroom (Ganoderma lucidum) extract in deep-fried meatballs: impact on heterocyclic aromatic amine formation.

BACKGROUND: The present research was conducted to investigate the impact of reishi mushroom extract (RME) on the formation of heterocyclic aromatic amines (HAAs) in meatballs. Sample preparations involved applying RME using either the spreading or addition method, with varying concentrations (0%, 0.25%, 0.5%, and 1% of RME), followed by deep-frying at temperatures of 150 and 190 °C for 3 min. RESULTS: The types and levels of HAAs varied based on the frying temperature, method of extract application, and the extract concentration. Notably, total HAA contents increased with rising the frying temperature (P < 0.01) and varied from undetectable levels to 4.91 ng g-1 across all analyzed meatballs. The addition method was more effective than the spreading method (P < 0.01), and among the concentrations tested 0.25% RME exhibited the highest efficacy in reducing total HAAs (P < 0.05). Furthermore, the addition method inhibited lipid oxidation more efficiently compared to the surface spreading method (P < 0.05). CONCLUSION: This study demonstrated that RME had mitigating effects on HAAs depending on the concentration and frying conditions in deep-fried meatball samples. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

High-throughput screening of the Saccharomyces cerevisiae genome for 2-amino-3-methylimidazo [4,5-f] quinoline resistance identifies colon cancer-associated genes.

Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cigarette smoke. However, few eukaryotic resistance genes have been identified. We used Saccharomyces cerevisiae (budding yeast) to identify genes that confer resistance to 2-amino-3-methylimidazo[4,5-f] quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. Deletion libraries expressing human CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 µM IQ for 5 or 10 generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. We identified 424 ORFs, including 337 genes of known function, in duplicate screens of the "humanized" collection for IQ resistance; resistance was further validated for a select group of 51 genes by growth curves, competitive growth, or trypan blue assays. Screens of the library not expressing human genes identified 143 ORFs conferring resistance to IQ per se. Ribosomal protein and protein modification genes were identified as IQ resistance genes in both the original and "humanized" libraries, while nitrogen metabolism, DNA repair, and growth control genes were also prominent in the "humanized" library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. Among DNA Repair and checkpoint genes, we identified those that function in postreplication repair (RAD18, UBC13, REV7), base excision repair (NTG1), and checkpoint signaling (CHK1, PSY2). These studies underscore the role of ribosomal protein genes in conferring IQ resistance, and illuminate DNA repair pathways for conferring resistance to activated IQ.

Heterocyclic aromatic amines in roasted chicken: Formation and prediction based on heating temperature and time.

The effects of chicken roasting temperature and time on the production of heterocyclic aromatic amines (HAAs) were investigated and an HAA prediction model based on heating conditions was established. Generally, the HAA content was significantly affected by the heating conditions in the roast chicken. Transportation of precursors from meat to skin, exposure of skin to high temperatures, and fat oxidation in the skin may result in higher HAAs than meat. Principal component analysis (PCA) showed that the effect of relatively high temperatures and long roasting times on HAAs was stronger than that of lower temperatures and shorter roasting times. In the prediction of HAA production, all regression correlation coefficient (R) values were close to one. The errors of 15 samples of experimental and predictive data were close to zero. Based on the results, backpropagation-artificial neural network (BP-ANN) has a high potential for predicting the production of HAAs under heating conditions.

Cyanidin and rutin inhibit the formation of heterocyclic aromatic amines in chemical modeling systems and smoked chicken drumsticks.

This study aims to evaluate the heterocyclic aromatic amine (HAA) formation inhibited by cyanidin and rutin in chemical modeling systems and smoked chicken drumsticks. In the PhIP and ß-carboline chemical modeling systems, cyanidin inhibited the PhIP, Harman, and Norharman formation while rutin inhibited the PhIP formation but promoted the Harman and Norharman formation compared with control sample (P < 0.05). A mechanistic investigation confirmed that inhibiting the PhIP formation by cyanidin was mainly through trapping phenylalanine, creatine, creatinine, glucose, phenylacetaldehyde, and an aldol condensation product. In the smoked chicken drumsticks, cyanidin inhibited the PhIP, Harman, and Norharman formation dose-dependently compared to the control sample (P < 0.05), with 2.0 % (w/v) cyanidin having the highest inhibitory effect. Moreover, cyanidin inhibited the formation of PhIP precursors and intermediates in the smoked chicken drumsticks. These results provide evidence for using phenolic compounds to reduce HAA formation in smoked meat products.

Understanding the Development of Heterocyclic Aromatic Amines in Fried Bacon and in the Remaining Oil after Pan-Frying in Five Different Vegetable Oils.

The physicochemical properties of five vegetable oils (peanut, corn, rapeseed, sunflower seed, and soybean) and their impact on the development of heterocyclic aromatic amines (HAAs) in pan-fried bacon and in the remaining oil were investigated. Corn oil led to the lowest total free amino acids (FAAs) contents and glucose content of fried bacon (p < 0.05) and rapeseed oil led to the lowest creatine content of fried bacon (p < 0.05). Bacon fried in corn oil had the highest HAA contents (p < 0.05). The total HAA contents of the oils after frying were lowest in rapeseed and soybean oils (p < 0.05). The type of vegetable oil used affected the color of the fried bacon but not the flavor and taste (p < 0.05). To reduce the HAA concentrations of fried bacon, the type of vegetable oil should be considered.

Heterocyclic aromatic amine contents and quality characteristics of bacon as influenced by NaCl concentration of brine.

Bacon usually have a high salt content. Excessive intake of salt could cause a harm to human health. Heterocyclic aromatic amines (HAAs) are carcinogenic and mutagenic heterocyclic compounds formed by the reactions of precursor substances at high temperature. This study investigated the influence of different levels of NaCl on the HAA contents and quality characteristics of bacon. Moisture, aw , L* value, b* value, thiobarbituric acid-reactive substance (TBARS), and carbonyl content increased significantly with a decrease in the NaCl concentration of the brine (p < 0.05). There were no significant differences between treatments for a* value, pH, creatine content, sensory redness, bitterness, or off-odor (p > 0.05). Sensory evaluation showed that saltiness in bacon increased significantly with increased NaCl concentrations in brine. The increased NaCl concentrations decreased the total HAAs in fried bacon (p < 0.05). Moreover, the nonpolar HAA contents in bacon were higher than the polar HAA contents; salt concentration mainly affected the nonpolar HAA content. In summary, salt content had a significant influence on the HAA content and the quality characteristic of bacon.

Comparison of the reducing effects of blueberry and propolis extracts on heterocyclic aromatic amines formation in pan fried beef.

This study was aimed to examine the effects of natural antioxidant-based marinades on the formation of heterocyclic aromatic amines (HAAs) in cooked beef. Samples were marinated overnight with different concentrations (0, 0.25, 0.5 and 1%) of blueberry or propolis extracts, and pan fried at 150 and 200 °C for a total of 10 min. Individual and total HAAs were detected at different levels according to the cooking temperature, extract type and concentration. Total HAA levels increased as the cooking temperature increased (P < 0.01), and ranged from non detectable levels to 38.27 ng/g across all samples analysed. Mitigation up to 61.2% and 100% in total HAA levels were achieved by propolis and blueberry extracts, respectively. The inhibitory effects of blueberry extract on total HAAs formation were significantly higher than propolis extract (P < 0.01). Thereby, this study showed that natural antioxidant-based marinades containing blueberry and propolis extracts had mitigating effects on HAAs formation in beef samples pan-fried at 150 and 200 °C.

Influence of Partial Replacements of NaCl by KCl on Quality Characteristics and the Heterocyclic Aromatic Amine Contents of Bacon.

The influence of partial replacements of NaCl by KCl (0, 10, 20, and 30%) on the heterocyclic aromatic amine (HAAs) contents and quality characteristics of bacon were investigated. The Na+ content, moisture, aw, pH, L* value, and sensory saltiness decreased and K+ content, a* value, and sensory bitterness increased significantly with increased substituting rates of NaCl by KCl (p < 0.05). There were no significant differences between the control and KCl substitution samples for the b* value, redness, and sensory off-odor (p > 0.05). The creatine content was not affected by the different KCl-substituting rates during the marinating process (p > 0.05), but it diminished in the smoking and frying processes (p < 0.05). The increase in the KCl-substituting rates increased the total heterocyclic aromatic amine (HAA) contents in fried bacon (p < 0.05). Moreover, the nonpolar HAA content in bacon was higher than the polar HAA content (p < 0.05). In summary, the partial replacement of NaCl by KCl increased the total HAA content and led to changes in bacon quality.

Effect of different types of smoking materials on the flavor, heterocyclic aromatic amines, and sensory property of smoked chicken drumsticks.

This study investigated the effect of different types of smoking materials on the flavor, heterocyclic aromatic amine (HAA) content, and sensory attributes of smoked chicken drumsticks. All smoked samples showed lower pH and L*-value and higher a*-value and b*-value than the control sample (P < 0.05), but no significant differences in water content and water activity (P > 0.05). The samples smoked with sucrose combined with pear-tree woodchips (SP) or green tea leaves (ST) had higher overall acceptability than other samples (P < 0.05). Smoking increased the total HAA content, and the ST sample exhibited the highest total HAA content (P < 0.05). A total of 54 volatile compounds was identified. Overall, SP and ST are suitable for smoked chicken considering the sensory properties, while S and SA are proper for smoked chicken considering the minimization of HAAs, which may provide a theory basis for the production of smoked chicken.

Selective Impedimetric Chemosensing of Carcinogenic Heterocyclic Aromatic Amine in Pork by dsDNA-Mimicking Molecularly Imprinted Polymer Film-Coated Electrodes.

Inspired by the easy intercalation of quinoxaline heterocyclic aromatic amines (HAAs) in double-stranded DNA (dsDNA), we synthesized a nucleobase-functionalized molecularly imprinted polymer (MIP) as the recognition unit of an impedimetric chemosensor for the selective determination of a 2-amino-3,7,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (7,8-DiMeIQx) HAA. HAAs are generated in meat and fish processed at high temperatures. They are considered to be potent hazardous carcinogens. The MIP film was prepared by potentiodynamic electropolymerization of a pre-polymerization complex of two adenine- and one thymine-substituted bis(2,2'-bithien-5-yl)methane functional monomer molecules with one 7,8-DiMeIQx template molecule, in the presence of the 2,4,5,2',4',5'-hexa(thiophene-2-yl)-3,3'-bithiophene cross-linking monomer, in solution. The as-formed MIP chemosensor allowed for the selective impedimetric determination of 7,8-DiMeIQx in the 47 to 400 µM linear dynamic concentration range with a limit of detection of 15.5 µM. The chemosensor was successfully applied for 7,8-DiMeIQx determination in the pork meat extract as a proof of concept.

Determination of creatine, creatinine, free amino acid and heterocyclic aromatic amine contents of plain beef and chicken juices.

In the present study, creatine, creatinine, free amino acid and heterocyclic aromatic amine (HAA) contents of plain beef and chicken juices were investigated. For this aim, the boned and boneless beef and chicken juices were obtained by boiling for 3 h at 100 °C without any additives in the laboratory. Creatine and creatinine content of the beef juices varied between 1.33-3.16 mg g-1 and 1.29-1.42 mg g-1, respectively, while creatine and creatinine content of the chicken juices varied between 0.98-1.63 mg g-1 and 0.89-1.30 mg g-1, respectively. The total free amino acid content in the beef juices and the chicken juices ranged between 14.61-20.65 mg 100 gdm-1 and 19.66-57.88 mg 100 gdm-1, respectively. None of the heterocyclic aromatic amines analyzed in the present study could be detected in the beef juices and the chicken juices. Therefore, the boned and boneless beef and chicken juice can be considered as safe from the standpoint of these HAAs and are suggested to use in the preparation of meals.

Effect of basil use in meatball production on heterocyclic aromatic amine formation.

Herein, the effects of basil usage in meatball production on various quality criteria and heterocyclic aromatic amine (HAA) formation were investigated. The use of basil at every rate caused a significant reduction in TBARS value compared to control group. Cooking caused an increase in pH and TBARS values. IQx, IQ, AαC and MeAαC compounds could not be detected, while various amounts of MeIQx, MeIQ, 7,8-DiMeIQx, 4,8-DiMeIQx and PhIP were determined in the samples. Total HAA contents were determined up to 1.61 ng g-1 and increasing of cooking temperature increased total HAA content, except for meatball with 1% basil. The reducing or enhancing effect of the use of basil in meatball production on the formation of HAA varied depending on the usage rate and cooking temperature. It was determined that even if 100 g of the meatballs containing 0.5% basil cooked at 250 °C whose total amount of HAA content was the highest, is eaten, the intake amount is far below 1 µg.

High-throughput quantification of eighteen heterocyclic aromatic amines in roasted and pan-fried meat on the basis of high performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry.

Triple quadrupole mass spectrometry has been the main technique for HAAs analysis in recent decade, while it requires extensive optimization of compound-dependent parameters. A novel method based on HPLC-Q-Orbitrap-HRMS was developed firstly for simultaneous determination of eighteen HAAs. Extraction and purification conditions were optimized and the developed method was validated in terms of linearity, accuracy and precision. Results indicated eighteen HAAs and two internal standards could be separated in 12 min using a gradient elution program. The full MS/dd-MS2 scan was adopted for analysis, which indicated favorable recoveries (71.3-114.8%) along with LODs and LOQs in the ranges of 0.02-0.6 and 0.05-2.0 µg/kg, respectively. Internal standards used for calibration could effectively reduce quantification errors produced by matrix effects. The validated method was successfully applied for HAAs analysis in roasted and pan-fried meat and was confirmed to be an alternative method when triple quadrupole mass spectrometry is absent in lab.

Is oven bag really advantageous in terms of heterocyclic aromatic amines and bisphenol-A? Chicken meat perspective.

Herein, the effects of oven bag use belong to different brands on heterocyclic aromatic amine (HAA) formation and bisphenol-A (BPA) migration in cooked chicken meats (breast and leg) were investigated. Samples were also analyzed in terms of some qualitative properties (fatty acid profile, water, fat, pH, TBARS, cooking loss). Both oven bag use and meat type had an effect on qualitative properties of the samples. Total HAA amount changed between 6.53 and 42.32 ng/g, and HAA content was higher in breast meat. Total BPA content in samples cooked with oven bag ranged between non-quantified to 63.78 ng/g. Oven bag use reduced the total HAA amount at the rate of 12 - 68.82%, while it caused the BPA migration depends on the brand. However, it can be noted that the HAA and BPA levels were not at a level to pose a risk to human health in any of the samples.

Evaluating the effects of temperature and time on heterocyclic aromatic amine profiles in roasted pork using combined UHPLC-MS/MS and multivariate analysis.

In this study, ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) combined with principal component analysis (PCA) were used to investigate the effects of process conditions on the profiles of carcinogenic and mutagenic heterocyclic aromatic amine (HAA) in the pork roasted at 175 °C, 200 °C, 225 °C and 250 °C for 10, 15, 20, 25, 30, 35 and 40 min. Twelve HAAs from four categories, including carboline (Norharman, Harman, and Phe-p-1), imidazopyridine (PhIP, 4'-OH-PhIP, DMIP, and 1,5,6-TMIP), imidazoquinoline (IQ, IQ [4,5-b], and MeIQ), and imidazoquinoxaline (MeIQx and 4,8-DiMeIQx), were detected, quantified and used to compose the HAA profiles in roasted pork. After being Analyzed by PCA, the distributions of HAA profiles from different temperature on the PCA score plot demonstrated that there are significant differences among the HAA profiles from different temperatures. The loading plot also showed that PhIP, 4'-OH-PhIP, IQ[4,5-b], and MeIQ were mainly responsible for the difference. The profiles from higher temperature distribute more scattered than the lower ones, illustrating that the time effects on the HAA profiles from higher temperature are stronger than the lower ones. Comparing the score and loading plots of different heating times, the diversities of the HAA profiles at different temperatures increased under prolonged heating because of the changingpyridines levels. The results of PCA that comparing the HAA from different categories displayed that the formation features of four categories HAAs were significantly differed because of their formation discrepancy under low temperatures and short-term roasting. Using HAA profiles as an entirety, these findings obtained in this study are more close to the real process of HAA formation in roasted pork, and make the complex effects of temperature and time on multiple HAA formations more simply to be concluded.

Heterocyclic aromatic amine level and quality characteristics of selected Harbin red sausages in the northern Chinese market.

The heterocyclic aromatic amine (HAA) level and quality characteristics of selected Harbin red sausages in the northern Chinese market and the possible differences between traditional sausages and conventional sausages were evaluated in this study. Four varieties of traditional sausages and four varieties of conventional sausages were selected. Compared to conventional sausages, traditional sausages had lower moisture content and higher hardness (P < 0.05). Twelve HAAs were evaluated and eight HAAs were detected. The total HAA content was as high as 360.73 ng/g. In particular, the contents of Norharman and Harman were much higher than those of the other HAAs (P < 0.05). Additionally, the HAA contents were higher in the traditional sausages than those in the conventional sausages (P < 0.05). Principal component analysis showed that traditional and conventional sausages had a good separation based on the quality characteristics and total HAA level. The results of this study will provide useful information on the industrial production of smoked meat products.

Potential carcinogenic heterocyclic aromatic amines (HAAs) in foodstuffs: Formation, extraction, analytical methods, and mitigation strategies.

During the heat treatment of proteinaceous food, heterocyclic aromatic amines (HAAs), a kind of strong mutagens/carcinogens are formed. HAAs can be classified into two major groups based on the heating temperature, which are thermic HAAs generally formed in 150 to 300 °C and pyrolytic HAAs produced above 300 °C. This review focuses on the formation mechanisms of HAAs and identifies different mechanisms of the formation of HAAs in foodstuffs. Moreover, an overview of the available extraction, purification methods, and instrumental analytical methods in the last two decades is shown to determine the HAAs in various foodstuffs. Finally, based on the factors that affect the formation of HAAs in heat-processed foodstuffs, such as the cooking method, food type, the recipe, and the content of substances with enhancing or inhibiting effects on the formation of HAAs, this review also highlights the most promising strategies for mitigating HAAs, which include adjusting cooking methods or process conditions, adding natural product extracts, antioxidants or other compounds, or reasonable selection of types of foodstuff. The review intends to provide a broad but comprehensive understanding of the formation, extraction, purification, analytical methods, and possible mitigation strategies for isolated and identified HAAs.

Neuromelanin formation exacerbates HAA-induced mitochondrial toxicity and mitophagy impairments.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is a major public health concern due in part to prevalence, debilitating symptoms, and links to environmental exposures. Much research has focused on environmental factors that may lead to dopaminergic neurotoxicity that occurs in PD. In the study of neuronal uptake and neurotoxicity, critical species differences have been observed. For example, neuromelanin is a molecule formed in part by the breakdown products of dopamine metabolism, along with lipid and protein components. Interestingly, human catecholaminergic neurons contain readily detectable amounts of neuromelanin, while rodent models form far lower levels of neuromelanin that is barely detectable. This discrepancy is potentially an important translational weakness. Recently, we showed that neuromelanin formation modulates heterocyclic aromatic amine (HAA)-induced neurotoxicity in cellular models. HAAs are dietary toxins that have primarily been studied as carcinogens, with emergent literature on selective neurotoxicity. The goal of the present study was to identify whether mitochondria in neuromelanin forming cells may be especially sensitive to HAAs. Here, we exposed galactose-supplemented SH-SY5Y cells to HAAs and tested mitochondrial function and mitophagy. The ectopic formation of neuromelanin was found to increase mitochondrial oxidative stress, decrease membrane potential, increase mitochondrial bioenergetic impairments, and impair mitophagy relative to HAA-treated cells that do not form neuromelanin. These results suggest that neuromelanin has a critical role in HAA toxicity and adverse effects on mitochondria. The data also further cement the need to conduct both mechanistic and risk assessment studies on PD-relevant neurotoxicity in models that form neuromelanin.

PhIP exposure in rodents produces neuropathology potentially relevant to Alzheimer's disease.

Alzheimer's disease (AD) is a public health crisis due to debilitating cognitive symptoms and lack of curative treatments, in the context of increasing prevalence. Thus, it is critical to identify modifiable risk factors. High levels of meat consumption may increase AD risk. Many toxins are formed during meat cooking such as heterocyclic aromatic amines (HAAs). Our prior studies have shown that HAAs produce dopaminergic neurotoxicity. Given the mechanistic and pathological overlap between AD and dopaminergic disorders we investigated whether exposure to 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), a prevalent dietary HAA formed during high-temperature meat cooking, may produce AD-relevant neurotoxicity. Here, C57BL/6 mice were treated with 100 or 200 mg/kg PhIP for 8 h or 75 mg/kg for 4 weeks and 16 weeks. PhIP exposure for 8 h produced oxidative damage, and AD-relevant alterations in hippocampal synaptic proteins, Amyloid-beta precursor protein (APP), and ß-Site amyloid precursor protein cleaving enzyme 1 (BACE1). PhIP exposure for 4 weeks resulted in an increase in BACE1. PhIP exposure for 16 weeks resulted in increased hippocampal oxidative damage, APP, BACE1, Aß aggregation, and tau phosphorylation. Quantification of intracellular nitrotyrosine revealed oxidative damage in cholinergic neurons after 8 h, 4 weeks and 16 weeks of PhIP exposure. Our study demonstrates that increase in oxidative damage, APP and BACE1 might be a possible mechanism by which PhIP promotes Aß aggregation. Given many patients with AD or PD exhibit neuropathological overlap, our study suggests that HAA exposure should be further studied for roles in mediating pathogenic overlap.